Actin is a widely expressed protein found in almost all eukaryotic cells. In humans, there are six different genes, which encode specific actin isoforms. Disease-causing mutations have been described for each of these, most of which are missense. Analysis of the position of the resulting mutated residues in the protein reveals mutational hotspots. Many of these occur in regions important for actin polymerization. We briefly discuss the challenges in characterizing the effects of these actin mutations, with a focus on cardiac actin mutations.

Over 1000 disease-causing missense mutations have been found in human β-cardiac, α-cardiac, embryonic and adult fast myosin 2a myosin heavy chains. Most of these are found in human β-cardiac myosin heavy chain. Mutations in β-cardiac myosin cause hypertrophic cardiomyopathy predominantly, whereas those in α-cardiac are associated with many types of heart disease, of which the most common is dilated cardiomyopathy. Mutations in embryonic and fast myosin 2a affect skeletal muscle function. This review provides a short overview of the mutations in the different myosin isoforms and their disease-causing effects. Keywords β-Cardiac myosin. α-Cardiac myosin. Embryonic myosin. Cardiac disease. Skeletal muscle disease. Missense mutation Abbreviations FHC Familial hypertrophic cardiomyopathy HCM Hypertrophic cardiomyopathy DCM Dilated cardiomyopathy LDM Laing distal myopathy MSM Myosin storage myopathy S1 Subfragment-1 (myosin motor domain) S2 Subfragment-2 LMM Light meromyosin (myosin tail) RLC Regulatory light chain ELC Essential light chain

Over 20 mutations in β-cardiac myosin heavy chain (β-MHC), expressed in cardiac and slow muscle fibers, cause Laing early-onset distal myopathy (MPD-1), a skeletal muscle myopathy. Most of these mutations are in the coiled-coil tail and commonly involve a mutation to a proline or a single-residue deletion, both of which are predicted to strongly affect the secondary structure of the coiled coil. To test this, we characterized the effects of two MPD-1 causing mutations: A1603P and K1617del in vitro and in cells. Both mutations affected secondary structure, decreasing the helical content of 15 heptad and light meromyosin constructs. Both mutations also severely disrupted the ability of glutathione S-transferase–light meromyosin fusion proteins to form minifilaments in vitro, as demonstrated by negative stain electron microscopy. Mutant eGFP-tagged β-MHC accumulated abnormally into the M-line of sarcomeres in cultured skeletal muscle myotubes. Incorporation of eGFP-tagged β-MHC into sarcomeres in adult rat cardiomyocytes was reduced. Molecular dynamics simulations using a composite structure of part of the coiled coil demonstrated that both mutations affected the structure, with the mutation to proline (A1603P) having a smaller effect compared to K1617del. Taken together, it seems likely that the MPD-1 mutations destabilize the coiled coil, resulting in aberrant myosin packing in thick filaments in muscle sarcomeres, providing a potential mechanism for the disease.

Dilated Cardiomyopathy is a common form of heart failure. Determining how this disease affects the structure and organization of cardiomyocytes in the human heart is important in understanding how the heart becomes less effective at contraction. Here we isolated and characterised Affimers (small non-antibody binding proteins) to Z-disc proteins ACTN2 (α-actinin-2), ZASP (also known as LIM domain binding protein 3 or LDB3) and the N-terminal region of the giant protein titin (TTN Z1-Z2). These proteins are known to localise in both the sarcomere Z-discs and the transitional junctions, found close to the intercalated discs that connect adjacent cardiomyocytes. We use cryosections of left ventricles from two patients diagnosed with end-stage Dilated Cardiomyopathy who underwent Orthotopic Heart Transplantation and were whole genome sequenced. We describe how Affimers substantially improve the resolution achieved by confocal and STED microscopy compared to conventional antibodies. We quantified the expression of ACTN2, ZASP and TTN proteins in two patients with dilated cardiomyopathy and compared them with a sex- and age-matched healthy donor. The small size of the Affimer reagents, combined with a small linkage error (the distance from the epitope to the dye label covalently bound to the Affimer) revealed new structural details in Z-discs and intercalated discs in the failing samples. Affimers are thus useful for analysis of changes to cardiomyocyte structure and organisation in diseased hearts.